The enzyme creatine kinase (CK; EC 2. 7. 3. 2) catalyzes the reversible phosphorylation of creatine in which adenosine-5'-triphosphate (ATP) serves as the donor: ##STR1## The forward reaction in which ATP is converted to adenosine-5'-diphosphate (ADP) is favored at about pH 9 whereas the reverse reaction is favored at about pH 7. The biological function of CK is the storage of high-energy creatine phosphate in the cell, and large quantities of the enzyme are present in skeletal muscle.
Chemically, CK is a dimer consisting of two molecular subunits designated as the M and B subunits which combine to give three isoenzymes: CK-BB, CK-MB and CK-MM. The three isoenzymes are located in the cytoplasm and each has a molecular weight of about 82,000 daltons. These isoenzymes can be separated by agarose gel electrophoresis in which the CK-BB isoenzyme migrates farthest toward the anode whereas the CK-MM isoenzyme migrates toward the cathode and the CK-MB isoenzyme migrates between the two.
The CK in the serum of the normal adult human consists mainly of the CK-MM isoenzyme with only trace quantities of the CK-MB. The CK-BB isoenzyme is not normally present in the serum at the detection limits of most CK assays. The detection of significant quantities of CK-MB in serum usually is indicative of acute myocardial infarction (AMI). However, CK-MB also has been found in the serum of patients with disorders other than AMI. Therefore, the CK-MB isoenzyme assay results need to be carefully interpreted by the medical profession. Nevertheless, most current assay methods are dedicated to the quantitation of CK-MB in AMI.
Various methods have been developed heretofore for the assay of CK, including spectrophotometric, colorimetric, fluorimetric, and coupled enzymatic methods.
In one typical coupled enzyme system, the reaction of creatine and ATP is initially catalyzed by CK to form creatine phosphate and ADP. This reaction is then coupled to two other enzyme reactions which employ phosphoenolpyruvate, reduced nicotinamide adenine dinucleotide (NADH) and the enzymes pyruvic kinase and lactate dehydrogenase. These reactions lead ultimately to the oxidation of NADH which is followed spectrophotometrically at 340 nm. This method was developed essentially by Tanzer and Gilvarg, J Biol Chem (1959) 234:3201-4, and modifications are described in U.S. Pat. No. 3,403,077.
Another coupled enzyme method is based on the reverse reaction in which creatine phosphate and ADP substrates react in the presence of CK to form creatine and ATP. The ATP generated serves in an auxiliary reaction to phosphorylate glucose in the presence of hexokinase (HK). The resulting glucose-6-phosphate (G-6-P) then becomes a substrate for the ultimate indicator reaction which is catalyzed by glucose-6-phosphate dehydrogenase (G-6-PDH) in the presence of nicotinamide adenine dinucleotide phosphate (NADP) to form 6-phosphogluconate and (NADPH). The production of NADPH is followed spectrophotometrically at 340 nm. This coupled enzyme system can be shown by the following series of equations: ##STR2##
The latter coupled enzyme system, first described by Nielsen and Ludvigsen and by Oliver, has been amplified by Rosalki, J Lab Clin Med (1967) 69:696-705, and further modifications are disclosed in U.S. Pat. Nos. 3,413,198; 3,485,724; 3,540,984; and 3,994,783.
Alternatively, the NADP and G-6-PDH in reaction 3, above, can be replaced with NAD and a G-6-PDH enzyme specific therefor, respectively, to produce NADH which can similarly be measured spectrophotemetrically. The presence of NADH also can be detected by other means. Thus, the substrate solution can include a dye which is reducible by NADH, thereby permitting the use of colorimetric procedures.
In another alternate procedure, reactions 2 and 3, above, can be omitted and the creatine in reaction 1 can be reacted with .alpha.-napthol and diacetyl to form a pink-colored complex.
The most common assay procedures for CK-MB consist of measuring the enzymatic activity of CK-MB following separation from other CK isoenzymes on the basis of differences in charge utilizing electrophoresis or ion-exchange, or by immunoinhibition or a combination of immunoinhibition and immunoprecipitation. Alternatively, the mass of CK-MB has been measured by immunoassay either using one antibody for the assay of B subunits (CK-MB plus CK-BB) or two-site antibody techniques. In the two-site antibody techniques, one antibody, for instance to the B subunit, is attached to a solid phase to extract the isoenzymes containing that subunit and, after washing, a labeled (enzyme or .sup.125 I) antibody to the other, in this case M, subunit is added.
In a recently developed typical example of the solid phase two-site antibody technique for assay of CK-MB (such as the Enzygnost immunoassay manufactured by Cal Biochem-Boehring, La Jolla, California), serum is added to tubes that are coated with antibodies specific for the CK-B subunit so as to bind CK-MB, CK-BB, and macro-CK-1 (CK-BB associated with immunoglobulin). After a wash step that removes the CK-MM isoenzyme and all other enzymes that do not contain a CK-B subunit, a second antibody (which is specific for the CK-M subunit and labeled with horseradish peroxidase enzyme) is added, thereby labeling only the CK-MB that remains in the assay tube. The substrate, urea peroxide, is then added. The concentration of the generated color product is then related to the concentration of CK-MB in the sample.
The most recently described procedures for quantitatively measuring the amount of CK-MB in sera thus are those based on an immunologic approach. See, for example, U.S. Pat. Nos. 4,260,678; 4,353,982; and 4,387,160. Monoclonal antibodies (as distinguished from polyclonal antibodies) to the B and M subunits of CK also have been developed for such assays and are described, for example, by Wurzburg and Strobel, J Clin Chem Clin Biochem (1981) 19:543-544; Morris and Head, FEBS Lett (1982) 145:163-168; Morris and Head, Biochem J (1983) 213:417-425; Jackson et al, Ibid (1983) 215:505-512. Assays for CK-MB using such antibodies have been further evaluated and reported by Jackson et al, Clin Chem (1984) 30:1157-1162; Sheehan and Haythorn, Ibid (1985) 31:160-161; Chan et al, Ibid (1985) 31:465-469; McBride et al, Ibid (1985) 31:1099-1100.
The prior assays have varying degrees of sensitivity to the other two isoenzymes of CK, namely CK-MM and CK-BB. In addition, interference by adenylate kinase (when CK activity is measured), by macro-CK-1 (CK associated with immunoglobulin) and by macro-CK-2 (mitochondrial CK) also have been encountered. Also, interference due to non-specific binding is a frequent problem in two-site assays (Boscato, L. M. et al., Clin Chem (1986) 32:1491-1495).